Cyclic peptide derivatives

ABSTRACT

Compounds of the formula I 
     
       
         cyclo-(Arg-X-Asp-R 1 )  I 
       
     
     in which 
     X is Gly, Ala or NH—NH—CO, 
     R 1  is a radical of the formula II                    
      and R 2 ,R 3  and R 4  have the meanings indicated in claim 1, 
     and their salts, 
     can be used as integrin inhibitors, in particular for the prophylaxis and treatment of disorders of the circulation, in thrombosis, cardiac infarct, coronary heart disorders, arteriosclerosis, in pathological processes which are supported or propagated by angiogenesis and in tumour therapy.

The invention relates to compounds of the formula I

cyclo-(Arg-X-Asp-R¹)  I

in which

X is Gly, Ala or NH—NH—CO,

where the amino acids mentioned can also be derivatized, and the aminoacid residues are linked to one another in a peptide-like manner via theα-amino and α-carboxyl groups,

R¹ is a radical of the formula II

 R²,R³,R⁴ in each case independently of one another are H, A, Ar, R⁵-Ar,Het or R⁵-Het,

A is alkyl having 1-6 C atoms,

Ar is phenyl which is unsubstituted or mono-, di- or trisubstituted byR⁷,R⁸ or R⁹, or unsubstituted naphthyl,

R⁵ is alkylene having 1-6 C atoms,

R⁶,R^(6′) in each case independently of one another are H, A, benzyl orphenyl,

R⁷,R⁸,R⁹ in each case independently of one another are R⁶, OR⁶, Hal,NO₂, NR⁶R⁶, NHCOR⁶, CN, NHSO₂R⁶, COOR⁶ or COR⁶,

Hal is F, Cl, Br or I and

Het is a mono- or binuclear heterocycle having 1 to 4 N, O, and/or Satoms, which can be unsubstituted or mono-, di- or trisubstituted byHal, A, NR⁶R^(6′), CN or NO₂,

where, providing the residues are optically active amino acids and aminoacid derivatives, both the D- and the L-forms are included,

and their salts.

Similar cyclic peptide compounds are disclosed, for example, in DE 43 10643 or EP 0 683 173.

The invention was based on the object of discovering novel compoundshaving valuable properties, in particular those which can be used forthe production of medicaments.

It has been found that the compounds of the formula I and their saltshave very valuable pharmacological properties together with goodtolerability. They act especially as integrin inhibitors, inhibiting, inparticular, the interactions of the α_(v)-, β₃- or β₅-integrin receptorswith ligands, such as, for example, the binding of fibrinogen to theβ₃-integrin receptor. The compounds exhibit particular efficacy in thecase of the integrins α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(IIb)β₃ and alsoα_(v)β₆ and α_(v)β₈.

This action can be detected, for example, by the method which isdescribed by J. W. Smith et al. in J. Biol. Chem. 265, 12267-12271(1990).

The dependency of the origin of angiogenesis on the interaction betweenvascular integrins and extracellular matrix proteins is described by P.C. Brooks, R. A. Clark and D. A. Cheresh in Science 264, 569-71 (1994).

The possibility of the inhibition of this interaction and thus for theinitiation of apoptosis (programmed cell death) of angiogenic vascularcells by a cyclic peptide is described by P. C. Brooks, A. M.Montgomery, M. Rosenfeld, R. A. Reisfeld, T.-Hu, G. Klier and D. A.Cheresh in Cell 79, 1157-64 (1994).

Compounds of the formula I which block the interaction of integrinreceptors and ligands, such as, for example, of fibrinogen on thefibrinogen receptor (glycoprotein IIb/IIIa), prevent, as GPIIb/IIIaantagonists, the spread of tumour cells by metastasis. This is confirmedby the following observations: The spread of tumour cells from a localtumour into the vascular system takes place through the formation ofmicroaggregates (microthrombi) by interaction of the tumour cells withblood platelets. The tumour cells are shielded by protection in themicroaggregate and are not recognized by the cells of the immune system.The microaggregates can fix to vascular walls, whereby a furtherpenetration of tumour cells into the tissue is facilitated. Since theformation of microthrombi is mediated by fibrinogen binding to thefibrinogen receptors on activated blood platelets, the GPIIa/IIIbantagonists can be regarded as effective metastasis inhibitors.

The compounds of the formula I can be employed as pharmaceutical activecompounds in human and veterinary medicine, in particular for theprophylaxis and/or therapy of thrombosis, myocardial infarct,arteriosclerosis, inflammations, apoplexy, angina pectoris, oncoses,osteolytic diseases such as osteoporosis, pathologically angiogenicdiseases such as, for example, inflammations, ophthalmological diseases,diabetic retinopathy, macular degeneration, myopia, ocularhistoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma,ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis,restenosis after angioplasty, viral infection, bacterial infection,fungal infection, in acute kidney failure and in wound healing forassisting the healing process.

The compounds of the formula I can be employed as substances havingantimicrobial activity in operations where biomaterials, implants,catheters or heart pacemakers are used. They have an antiseptic effecthere. The efficacy of the antimicrobial activity can be demonstrated bythe method described by P.Valentin-Weigund et al., in Infection andImmunity, 2851-2855 (1988).

Since the compounds of the formula I are inhibitors of fibrinogenbinding and thus ligands of the fibrinogen receptors on blood platelets,they can be used as diagnostics for the detection and localization ofthrombi in the vascular system in vivo, provided they are substituted,for example, by a radioactive or Uv-detectable radical.

As inhibitors of fibrinogen binding, the compounds of the formula I canalso be used as efficacious medicaments for the study of the metabolismof blood platelets in different activation stages or of intracellularsignal mechanisms of the fibrinogen receptor. The detectable unit of a“label” to be incorporated, e.g. an isotope labelling by ³H, allows themechanisms mentioned to be investigated, after binding to the receptor.

The abbreviations of amino acid residues mentioned above and below standfor the radicals of the following amino acids:

Ala Alanine AMP Aminomethylphenyl residue Asn Asparagine Asp Asparticacid Arg Arginine Cys Cysteine Gln Glutamine Glu Glutamic acid GlyGlycine His Histidine homo-Phe homo-Phenylalanine Ile Isoleucine LeuLeucine Lys Lysine Met Methionine Nle Narleucine Orn Ornithine PhePhenylalanine Phg Phenylglycine 4-Hal-Phe 4-Halaphenylalanine ProProline Ser Serine Thr Threonine Trp Tryptophan Tyr Tyrosine Val Valine.

The 3-AMP radical has the following structure:

The following radicals have the meanings below:

Ac Acetyl BOC tert-Butoxycarbonyl CBZ or Z Benzyloxycarbonyl DCCIDicyclohexylcarbodiimide DMF Dimethylformamide EDCIN-Ethyl-N,N′-dimethylaminopropyl) carbodiimide Et Ethyl FCAFluoresceincarboxylic acid Fmoc 9-Fluorenylmethoxycarbonyl HOBt1-Hydroxybenzotriazole Me Methyl MBHA 4-Methylbenzhydrylamine Mtr4-Methoxy-2,3,6-trimethylphenylsulfonyl HONSu N-Hydroxysuccinimide OBzlBenzyl ester OtBu tert-Butyl ester Oct Octanoyl OMe Methyl ester OEtEthyl ester POA Phenoxyacetyl Sal Salicyloyl TFA Trifluoroacetic acidTrt Trityl (Triphenylmethyl).

Provided the abovementioned amino acids can occur in a number ofenantiomeric forms, all these forms and also their mixtures (e.g. theDL-forms) are included above and below, e.g. as a constituent of thecompounds of the formula I. Furthermore, the amino acids, e.g. as aconstituent of compounds of the formula I, can be provided withappropriate protective groups known per se.

In the compounds according to the invention, so-called prodrugderivatives are also included, i.e. compounds of the formula I modifiedwith, for example, alkyl or acyl groups, sugars or oligopeptides, whichare rapidly cleaved in the body to give the active compounds accordingto the invention.

These also include biodegradable polymer derivatives of the compoundsaccording to the invention, such as is described, for example, in Int.J. Pharm. 115, 61-67 (1995).

Amino acids whose configuration is not specifically indicated have the(S)- or (L)-configuration.

The invention further relates to a process for the preparation ofcompounds of the formula I according to claim 1, and their salts,characterized in that

(a) a compound of the formula III

H—Z—OH  III

 in which

Z is -Arg-X-Asp-R¹-

—X-Asp-R¹-Arg-

—Asp-R¹-Arg-X- or

—R¹-Arg-X-Asp,

and X and R¹ have the meanings indicated in Claim 1,

or a reactive derivative of a compound of the formula II is treated witha cyclizing agent, or

b) a compound of the formula I is liberated from one of its functionalderivatives by treating with a solvolysing or hydrogenolysing agent,

and/or in that a basic or acidic compound of the formula I is convertedinto one of its salts by treating with an acid or base.

Above and below, the radicals X, R¹,R² R³ and R⁴ have the meaningsindicated in the formulae I, II and III, if not expressly statedotherwise.

In the above formulae, alkyl is preferably methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, and further alsopentyl, 1-, 2- or 3-methylbutyl, 1,1-, 1,2- or 2,2-dimethylpropyl,1-ethylpropyl, hexyl, 1-, 2-, 3- or 4-methylpentyl, 1,1-, 1,2-, 1,3-,2,2-, 2,3- or 3,3-dimethylbutyl, 1- or 2-ethylbutyl,1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, 1,1,2- or1,2,2-trimethylpropyl.

R² and R³ are, in each case independently of one another, preferably,for example, H or A, and further also Ar or R⁵-Ar. R⁴ is preferably, forexample, H, A, Ar or R⁵-Ar, and further also Het or R⁵-Het. If R⁴ isalkyl, a methylene group present in the alkyl chain can also be replacedby N, O or S.

Alkylene is preferably methylene, ethylene, propylene, butylene,pentylene or hexylene. R⁵-Ar is preferably benzyl or phenethyl.

The amino acids and amino acid residues mentioned can also bederivatized, the N-methyl, N-ethyl, N-propyl, N-benzyl or C_(α)-methylderivatives being preferred. Derivatives of Asp and Glu are additionallypreferred, in particular the methyl, ethyl, propyl, butyl, tert-butyl,neopentyl or benzyl esters of the side chain carboxyl groups, andfurther also derivatives of Arg, which can be substituted on the—NH—C(═NH)—NH₂ group by an acetyl, benzoyl, methoxycarbonyl orethoxycarbonyl radical.

R⁶ is preferably, for example, H, methyl or ethyl, and further benzyl orphenyl. OR⁶ is preferably, for example, hydroxyl or methoxy. COR⁶ isalkanoyl and is preferably formyl, acetyl, propionyl, butyryl, pentanoylor hexanoyl.

Ar is unsubstituted, preferably—as indicated—monosubstituted phenyl,specifically preferably phenyl, o-, m-, or p-tolyl, o-, m- orp-ethylphenyl, o-, m- or p-propylphenyl, o-, m- or p-isopropylphenyl,o-, m- or p-tert-butylphenyl, o-, m- or p-trifluoromethylphenyl, o-, m-or p-hydroxyphenyl, o-, m- or p-nitrophenyl, o-, m- or p-aminophenyl,o-, m- or p-(N-methylamino)phenyl, o-, m- or p-acetamidophenyl, o-, m-or p-(trifluoromethoxy)phenyl, o-, m- or p-cyanophenyl, o-, m- orp-methoxyphenyl, o-, m- or p-ethoxyphenyl, o-, m- or p-carboxyphenyl,o-, m- or p-methoxycarbonylphenyl, o-, m- or p-ethoxycarbonylphenyl, o-,m- or p-benzyloxycarbonylphenyl, o-, m- or p-(carboxymethyloxy)phenyl,o-, m- or p-(methoxycarbonylmethyloxy)phenyl, o-, m- orp-(methoxycarbonylethyloxy)phenyl, o-, m- orp-(N,N-dimethylamino)phenyl, o-, m- or p-(N-ethylamino)phenyl, o-, m- orp-(N,N-diethylamino)phenyl, o-, m- or p-fluorophenyl, o-, m- orp-bromophenyl, o-, m- or p-chlorophenyl, o-, m- orp-(difluoromethoxy)phenyl, o-, -m- or p-(fluoromethoxy)phenyl, o-, m- orp-formylphenyl, o-, m- or p-acetylphenyl, o-, m- or p-propionylphenyl,o-, m- or p-butyrylphenyl, o-, m- or p-pentanoylphenyl, o-, m- orp-(phenylsulfonamidocarbonyl)phenyl, o-, m- or p-phenoxyphenyl, o-, m-or p-methylthiophenyl, o-, m- or p-methylsulfinylphenyl, o-, m- orp-methylsulfonylphenyl or naphthyl.

Het is preferably 2- or 3-furyl, 2- or 3-thienyl, 1-, 2- or 3-pyrrolyl,1-, 2, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 2-, 4- or5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or5-isothiazolyl, 2-, 3- or 4-pyridyl, 2-, 4-, 5- or 6-pyrimidinyl,furthermore preferably 1,2,3-triazol-1-, -4- or -5-yl, 1,2,4-triazol-1-,-3- or 5-yl, 1- or 5-tetrazolyl, 1,2,3-oxadiazol-4- or -5-yl,1,2,4-oxadiazol-3- or -5-yl, 1,3,4-thiadiazol-2- or -5-yl,1,2,4-thiadiazol-3- or -5-yl, 1,2,3-thiadiazol-4- or -5-yl, 2-, 3-, 4-,5- or 6-2H-thiopyranyl, 2-, 3- or 4-4-H-thiopyranyl, 3- or4-pyridazinyl, pyrazinyl, 2-, 3-, 4-, 5- 6- or 7-benzofuryl, 2-, 3-, 4-,5-, 6- or 7-benzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 1-, 2-,4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 2-, 4-,5-, 6- or 7-benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisoxazolyl, 2-, 4-,5-, 6- or 7-benzothiazolyl, 2-, 4-, 5-, 6- or 7-benzisothiazolyl, 4-,5-, 6- or 7-benz-2,1,3-oxadiazolyl, 2-, 3-, 4-, 5-, 6-, 7- or8-quinolyl, 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolyl, 3-, 4-, 5-, 6-, 7-or 8-cinnolinyl, 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl. The heterocyclicradicals can also be partially or completely hydrogenated.

Het can thus, for example, also be 2,3-dihydro-2-, -3-, -4- or -5-furyl,2,5-dihydro-2-, -3-, -4- or 5-furyl, tetrahydro-2- or -3-furyl,1,3-dioxolan-4-yl, tetrahydro-2- or -3-thienyl, 2,3-dihydro-1-, -2-,-3-, -4- or -5-pyrrolyl, 2,5-dihydro-1-, -2-, -3-, -4- or -2- or-4-imidazolyl, 2,3-dihydro-1-, -2-, -3-, -4- or -5-pyrazolyl,tetrahydro-1-, -3- or -4-pyrazolyl, 1,4-dihydro-1-, -2-, -3- or-4-pyridyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5- or -6-pyridyl, 1-,2-, 3- or 4-piperidinyl, 2-, 3- or 4-morpholinyl, tetrahydro-2-, -3- or-4-pyranyl, 1,4-dioxanyl, 1,3-dioxan-2-, -4- or -5-yl, hexahydro-1-, -3-or -4-pyridazinyl, hexahydro-1-, -2-, -4- or -5-pyrimidinyl, 1-, 2- or3-piperazinyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5-, -6-, -7- or-8-quinolyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5-, -6-, -7- or-8-isoquinolyl.

Amino protective group is preferably acetyl, propionyl, butyryl,phenylacetyl, benzoyl, toluyl, POA, methoxycarbonyl, ethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, BOC, 2-iodethoxycarbonyl, CBZ(“carbobenzoxy”), 4-methoxybenzyloxycarbonyl, FMOC, Mtr or benzyl.

The compounds of the formula I can have one or more chiral centres andtherefore occur in various stereoisomeric forms. The formula I includesall these forms.

Accordingly, the invention relates in particular to those compounds ofthe formula I in which at least one of the radicals mentioned has one ofthe preferred meanings indicated above. Some preferred groups ofcompounds can be expressed by the following subformulae Ia to Ie, whichcorrespond to the formula I and in which the radicals not described ingreater detail have the meaning indicated in formula I, but in which

in a) R²,R³ in each case independently of one another are H or A,

R⁴ is H, A, Ar, R⁵-Ar, Het or R⁵-Het and

R⁶,R^(6′) are H or A;

in b) R²,R³ in each case independently of one another are H or A,

R⁴ is H, A, Ar, R⁵-Ar, Het or R⁵-Het

R⁶,R^(6′) are H or A and

Ar is phenyl which is unsubstituted or monosubstituted by R⁷;

in c) R²,R³ in each case independently of one another are H or A,

R⁴ is H, A, Ar, R⁵-Ar, Het or R⁵-Het,

R⁶,R^(6′) are H or A,

Ar is phenyl which is unsubstituted or monosubstituted by R⁷ and

Het is a mononuclear aromatic or saturated heterocycle having 1 or 2 Nor O atoms, which can be unsubstituted or mono- or disubstituted by Hal,A, NR⁶R^(6′), CN or NO₂;

in d) R²,R³ in each case independently of one another are H or A,

R⁴ is H, A, Ar or R⁵-Ar,

R⁶,R^(6′) are H or A and

Ar is phenyl which is unsubstituted or monosubstituted by R⁷;

in e) X is Gly or Ala

R²,R³ in each case independently of one another are H or A,

R⁴ is H, A, Ar or R⁵-Ar,

R⁶,R^(6′) are H or A and

Ar is phenyl which is unsubstituted or monosubstituted by R⁷.

The compounds of the formula I and also the starting substances fortheir preparation are otherwise prepared by methods known per se, suchas are described in the literature (e.g. in the standard works such asHouben-Weyl, Methoden der organischen Chemie [Methods of OrganicChemistry], Georg-Thieme Verlag, Stuttgart), namely under reactionconditions which are known and suitable for the reactions mentioned. Usecan also be made in this case of variants which are known per se, butnot mentioned here in greater detail.

The starting substances, if desired, can also be formed in situ, suchthat they are not isolated from the reaction mixture, but immediatelyreacted further to give the compounds of the formula I.

Compounds of the formula I can preferably be obtained by cyclization ofcompounds of the formula III under the conditions of a peptidesynthesis. The reaction is expediently carried out here according tocustomary methods of peptide synthesis, as are described, for example,in Houben-Weyl, 1.c., Volume 15/II, pages 1 to 806 (1974).

The reaction preferably takes place in the presence of a dehydratingagent, e.g. of a carbodiimide such as DCCI or EDCI, and further, forexample, propanephosphonic anhydride (cf. Angew. Chem. 92, 129 (1980)),diphenylphosphoryl azide or2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline, in an inert solvent,e.g. a halogenated hydrocarbon such as dichloromethane, an ether such astetrahydrofuran or dioxane, an amide such as DMF or dimethylacetamide, anitrile such as acetonitrile, in dimethyl sulfoxide or in the presenceof mixtures of these solvents, at temperatures between approximately −10and 40, preferably between 0 and 30°. In order to promote intramolecularcyclization before intermolecular peptide bonding, it is expedient towork in dilute solutions. The reaction time, depending on the conditionsused, is between a few minutes and 14 days.

Instead of compounds of the formula III, derivatives of compounds of theformula III, preferably a preactivated carboxylic acid, or a carboxylicacid halide, a symmetrical or mixed anhydride or an active ester canalso be employed. Radicals of this type for the activation of thecarboxyl group in typical acylation reactions are described in theliterature (e.g. in the standard works such as Houben-Weyl, Methoden derorganischen Chemie [Methods of Organic Chemistry], Georg-Thieme Verlag,Stuttgart).

Activated esters are expediently formed in situ, e.g. by addition ofHOBt or N-hydroxysuccinimide.

As a rule, the reaction is carried out in an inert solvent, using acarboxylic acid halide in the presence of an acid-binding agent,preferably of an organic base such as triethylamine, dimethylaniline,pyridine or quinoline. The addition of an alkali metal or alkaline earthmetal hydroxide, carbonate or bicarbonate or of another salt of a weakacid of the alkali metals or alkaline earth metals, preferably ofpotassium, sodium, calcium or caesium, may also be favourable.

As a rule, the starting substances of the formula III are new. They canbe prepared by known methods of peptide synthesis.

The compounds of the formula I can further be obtained by liberatingthem from their functional derivatives by solvolysis, in particularhydrolysis, or by hydrogenolysis.

Preferred starting substances for solvolysis or hydrogenolysis are thosewhich, instead of one or more free amino and/or hydroxyl groups, containcorresponding protective amino and/or hydroxyl groups, preferably thosewhich, instead of an H atom which is bonded to an N atom, carry an aminoprotective group, e.g. those which correspond to the formula I, butinstead of an NH₂ group contain an NHR′ group (in which R′ is an aminoprotective group, e.g. BOC or CBZ).

Starting substances are further preferred which, instead of the H atomof a hydroxyl group, carry a hydroxyl protective group, e.g. those whichcorrespond to the formula I, but instead of a hydroxyphenyl groupcontain an R″O-phenyl group (in which R″ is a hydroxyl protectivegroup).

A number of—identical or different—protected amino and/or hydroxylgroups can also be present in the molecule of the starting substance. Ifthe protective groups present differ from one another, they can beselectively removed in many cases.

The expression “amino protective group” is generally known and relatesto groups which are suitable for protecting (for blocking) an aminogroup from chemical reactions, but which are easily removable after thedesired chemical reaction has been carried out at other positions in themolecule. Typical groups of this type are, in particular, unsubstitutedor substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since theamino protective groups are removed after the desired reaction (orreaction sequence), their nature and size is otherwise not critical;however, those having 1-20, in particular 1-8 C atoms, are preferred.The expression “acyl group” is to be interpreted in the widest sense inconnection with the present process. It includes acyl groups derivedfrom aliphatic, araliphatic, aromatic or heterocyclic carboxylic acidsor sulfonic acids, such as, in particular, alkoxycarbonyl,aryloxycarbonyl and especially aralkoxycarbonyl groups. Examples of acylgroups of this type are alkanoyl such as acetyl, propionyl, butyryl;aralkanoyl such as phenylacetyl; aroyl such as benzoyl or toluyl;aryloxyalkanoyl such as POA; alkoxycarbonyl such as methoxycarbonyl,ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC,2-iodoethoxy-carbonyl; aralkyloxycarbonyl such as CBZ (“carbobenzoxy”),4-methoxybenzyloxycarbonyl, FMOC; arylsulfonyl such as Mtr. Preferredamino protective groups are BOC and Mtr, and further CBZ, Fmoc, benzyland acetyl.

The expression “hydroxyl protective group” is likewise generally knownand relates to groups which are suitable for protecting a hydroxyl groupfrom chemical reactions, but which are easily removable after thedesired chemical reaction has been carried out at other positions in themolecule. Typical of such groups are the abovementioned unsubstituted orsubstituted aryl, aralkyl or acyl groups, and further also alkyl groups.The nature and size of the hydroxyl protected groups is not critical,since they are removed again after the desired chemical reaction orreaction sequence; groups having 1-20, in particular 1-10 C atoms, arepreferred. Examples of hydroxyl protective groups are, inter alia,benzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, benzyland tert-butyl being particularly preferred. The COOH groups in asparticacid and glutamic acid are preferably protected in the form of theirtert-butyl esters (e.g. Asp(OBut)).

The liberation of the compounds of the formula I from their functionalderivatives is carried out—depending on the protective group used—forexample, with strong acids, expediently with TFA or perchloric acid, butalso with other strong inorganic acids such as hydrochloric acid orsulfuric acid, strong organic carboxylic acids such as trichloroaceticacid or sulfonic acids such as benzene- or p-toluenesulfonic acid. Thepresence of an additional inert solvent is possible, but not alwaysnecessary. Suitable inert solvents are preferably organic solvents, forexample carboxylic acids such as acetic acid, ethers such astetrahydrofuran or dioxane, amides such as DMF, halogenated hydrocarbonssuch as dichloromethane, and further also alcohols such as methanol,ethanol or isopropanol, and also water. Mixtures of the abovementionedsolvents are also possible. TFA is preferably used in an excess withoutaddition of a further solvent, perchloric acid in the form of a mixtureof acetic acid and 70% strength perchloric acid in the ratio 9:1. Thereaction temperatures for the cleavage are expediently betweenapproximately [lacuna] and approximately 50°; the reaction is preferablycarried out between 15 and 30° (room temperature).

The groups BOC, OBut and Mtr can be removed, for example, preferablyusing TFA in dichloromethane or using approximately 3 to 5N HCl indioxane at 15-30°; the FMOC group using an approximately 5 to 50%solution of dimethylamine, diethylamine or piperidine in DMF at 15-30°.

The trityl group is employed for the protection of the amino acidshistidine, asparagine, glutamine and cysteine. The cleavage is carriedout, depending on the desired final product, using TFA/10% thiophenol,the trityl group being removed from all amino acids mentioned; whenusing TFA/anisole or TFA/thioanisole, the trityl group is removed onlyHis, Asn and Gln, while it remains on the Cys side chain.

Hydrogenolytically removable protective groups (e.g. CBZ or benzyl) canbe removed, for example, by treating with hydrogen in the presence of acatalyst (e.g. of a noble metal catalyst such as palladium, expedientlyon a support such as carbon). Suitable solvents here are those indicatedabove, in particular, for example, alcohols such as methanol or ethanolor amides such as DMF. As a rule, the hydrogenolysis is carried out attemperatures between approximately 0 and 100° and pressures betweenapproximately 1 and 200 bar, preferably at 20-30° C. and 1-10 bar.Hydrogenolysis of the CBZ group takes place, for example, readily on 5to 10% Pd/C in methanol or with ammonium formate (instead of hydrogen)on Pd/C in methanol/DMF at 20-30°.

A base of the formula I can be converted into the associated acidaddition salt using an acid, for example by reaction of equivalentamounts of the base and of the acid in an inert solvent such as ethanoland subsequent evaporation. For this reaction, suitable acids are inparticular those which yield physiologically acceptable salts. Thusinorganic acids can be used, e.g. sulfuric acid, nitric acid, hydrohalicacids such as hydrochloric acid or hydrobromic acid, phosphoric acids,such as orthophosphoric acid, sulfamic acid, and further organic acids,in particular aliphatic, alicyclic, araliphatic, aromatic orheterocyclic mono- or polybasic carboxylic, sulfonic or sulfuric acids,e.g. formic acid, acetic acid, propionic acid, pivalic acid,diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaricacid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid,gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid,methane- or ethanesulfonic acid, ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonicacid, naphthalenemono- and disulfonic acids, and laurylsulfuric acid.Salts with physiologically unacceptable acids, e.g. picrates, can beused for the isolation and/or purification of the compounds of theformula I.

On the other hand, an acid of the formula I can be converted into one ofits physiologically acceptable metal or ammonium salts by reaction witha base. Possible salts here are in particular the sodium, potassium,magnesium, calcium and ammonium salts, and also substituted ammoniumsalts, e.g. the dimethyl, diethyl or diisopropylammonium salts,monoethanol-, diethanol- or diisopropylammonium salts, cyclohexyl- ordicyclohexylammonium salts, dibenzylethylenediammonium salts, andfurthermore, for example, salts with arginine or lysine.

The invention further relates to the use of the compounds of the formulaI and/or their physiologically acceptable salts for the production ofpharmaceutical preparations, in particular by a non-chemical route. Inthis context, they can be brought into a suitable dosage form togetherwith at least one solid, liquid and/or semisolid excipient or auxiliaryand, if appropriate, in combination with one or more further activecompounds.

The invention further relates to pharmaceutical preparations comprisingat least one compound of the formula I and/or one of its physiologicallyacceptable salts.

These preparations can be used as medicaments in human or veterinarymedicine. Suitable excipients are organic or inorganic substances whichare suitable for enteral (e.g. oral) or parenteral administration,topical application or for administration in the form of an inhalationspray and do not react with the novel compounds, for example water,vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols,glycerol triacetate, gelatine, carbohydrates such as lactose or starch,magnesium stearate, talc and petroleum jelly. In particular, tablets,pills, coated tablets, capsules, powders, granules, syrups, juices ordrops are used for oral administration, suppositories are used forrectal administration, solutions, preferably oily or aqueous solutions,and also suspensions, emulsions or implants are used for parenteraladministration, and ointments, creams or powders are used for topicalapplication. The novel compounds can also be lyophilized and thelyophilizates obtained used, for example, for the production ofinjection preparations. The preparations indicated can be sterilizedand/or can contain auxiliaries such as lubricants, preservatives,stabilizers and/or wetting agents, emulsifiers, salts for affecting theosmotic pressure, buffer substances, colorants, flavourings and/or oneor more other active compounds, e.g. one or more vitamins.

For administration as an inhalation spray, sprays can be used whichcontain the active compound either dissolved or suspended in apropellant gas or propellant gas mixture (e.g. CO₂ orchlorofluorohydrocarbons). The active compound is expediently used herein micronized form, it being possible for one or more additionalphysiologically tolerable solvents to be present, e.g. ethanol.

Inhalation solutions can be administered with the aid of customaryinhalers.

The compounds of the formula I and their physiologically acceptablesalts can be used as integrin inhibitors in the control of illnesses, inparticular of thromboses, cardiac infarct, coronary heart disorders,arteriosclerosis, tumours, osteoporosis, inflammations and infections.

The compounds of the formula I according to claim 1 and/or theirphysiologically acceptable salts are also used in pathological processeswhich are supported or propagated by angiogenesis, in particular intumours or rheumatoid arthritis.

In this case, the substances according to the invention can as a rule beadministered in analogy to other known, commercially available peptides,but in particular in analogy to the compounds described in U.S. Pat. No.4,472,305, preferably in doses of between approximately 0.05 and 500 mg,in particular between 0.5 and 100 mg per dose unit. The daily dose ispreferably between approximately 0.01 and 2 mg/kg of body weight. Thespecific dose for each patient depends, however, on all sorts offactors, for example on the efficacy of the specific compound employed,on the age, body weight, general state of health, sex, on the diet, onthe time and route of administration, on the excretion rate,pharmaceutical combination and severity of the particular disorder towhich the therapy relates. Parenteral administration is preferred.

The compounds of the formula I can further be used as integrin ligandsfor the preparation of columns for affinity chromatography for thepreparation of integrins in pure form.

The ligand, i.e. a compound of the formula I, is in this case covalentlycoupled to a polymeric support via an anchor function, e.g. the carboxylgroup of Asp.

Suitable polymeric support materials are the polymeric solid phasesknown per se in peptide chemistry, preferably having hydrophilicproperties, for example crosslinked polysugars such as cellulose,Sepharose or Sephadex®, acrylamides, polymers based on polyethyleneglycol or Tentakel polymers®.

The preparation of the materials for affinity chromatography forintegrin purification is carried out under conditions such as arecustomary and known per se for the condensation of amino acids.

The compounds of the formula I contain one or more chiral centres andcan therefore be present in racemic or in optically active form.Racemates obtained can be separated into the enantiomers mechanically orchemically by methods known per se. Preferably, diastereomers are formedfrom the racemic mixture by reaction with an optically active resolvingagent. Suitable resolving agents are, for example, optically activeacids, such as the D- and L-forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. An enantiomer separation with the aid of acolumn packed with an optically active resolving agent (e.g.dinitrobenzyolphenylglycine) is also advantageous; suitable eluents are,for example, a mixture of hexane/isopropanol/acetonitrile, e.g. in thevolume ratio 82:15:3.

Of course, it is also possible to obtain optically active compounds ofthe formula I by the methods described above by using startingsubstances which are already optically active.

Above and below, all temperatures are indicated in ° C. In the followingexamples, “customary working up” means: if necessary, water is added,the mixture is adjusted, if necessary, to a pH of between 2 and 10depending on the constitution of the final product, and extracted withethyl acetate or dichloromethane, the organic phase is separated off,dried over sodium sulfate and evaporated, and the residue is purified bychromatography on silica gel and/or by crystallization. R_(f) values onsilica gel; eluents: ethyl acetate/methanol 9:1. RT=retention time(minutes) on HPLC in the following systems:

[A]

Column: Lichrosorb®RP 18 (250×4; 5 μm);

Eluent A: 0.1% TFA in water

Eluent B: 0.1% TFA in 90% acetonitrile, 10% water

Flow rate: 1 ml/min

Gradient: 20-95% B/50 min

Detection at 215 nm.

The diastereomers are preferably separated under the conditionsindicated.

Mass spectrometry (MS): FAB (Fast Atom Bombardment) (M+H)⁺

EXAMPLE 1

Equimolar amounts of methyl (R,S)-2-bromo-2-phenylacetate and3-hydroxymethylaniline are reacted to give methylN-(3-hydroxymethylphenyl)aminophenyl-acetate. By reaction with thionylchloride to give methyl N-(3-chloromethylphenyl)aminophenylacetate andsubsequent reaction with sodium azide, methylN-(3-azidomethylphenyl)aminophenylacetate (“A”) is obtained. A solutionof 9.2 g of “A” in 350 ml of ethyl acetate is hydrogenated for 35minutes in the presence of 1 g of Pd/C (5%). After removing the catalystand the solvent, methyl N-(3-aminomethylphenyl)aminophenyl-acetate)(“B”) is obtained as an oil, RT 19.5; FAB 271.

By reaction of “B” with benzyl anhydride, methylN-(3-benzyloxycarbonylaminomethyl-phenyl)aminophenylacetate is obtained,which is then hydrolyzed in KOH/methanol to giveN-(3-benzyloxycarbonylaminomethylphenylacetic acid(=N-(Z-3-AMP)-aminophenylacetic acid). By reaction with 1 equivalent ineach case of H-Arg(Mtr)-Gly-OtBu, DCCI and HOBt in dichloromethane,Z-3-AMP-Phg-Arg(Mtr)-Gly-OtBu is obtained. The removal of theZ-protective group is carried out as described above by catalytichydrogenation; subsequent peptide coupling withBOC-Asp(OBzl)-3-AMP-Phg-Arg(Mtr)-Gly-OtBu. After removal of the BOCprotective group and of the tert-butyl ester in HCl/dioxane,H-Asp(OBzl)-3-AMP-Phg-Arg(Mtr)-Gly-OH- is obtained, and, aftercyclization, the compound

Cyclo-(Asp(OBzl)-3-AMP-Phg-Arg(Mtr)-Gly). After hydrolysis of the ester,removal of the Mtr protective group in 98% trifluoroacetic acid,purification and separation by means of HPLC,

cyclo-(Asp-3-AMP-L-Phg-Arg-Gly) and

cyclo-(Asp-3-AMP-D-Phg-Arg-Gly) are obtained.

The two compounds are characterized as follows:

RT 15.5 FAB 567 and RT 12.5 FAB 567, the assignment for the twodiastereomers being open to question.

Analogously, the following are obtained starting from2-bromo-3-methylbutyric acid

cyclo-(Asp-3-AMP-L-Val-Arg-Gly) (SEQ ID NO: 1) and

cyclo-(Asp-3-AMP-D-Val-Arg-Gly), from 2-bromoacetic acid

cyclo-(Asp-3-AMP-Gly-Arg-Gly) (SEQ ID NO: 3) and from2-bromo-3-phenylpropionic acid

cyclo-(Asp-3-AMP-L-Phe-Arg-Gly) (SEQ ID NO: 2) and

cyclo-(Asp-3-AMP-D-Phe-Arg-Gly).

The following examples relate to pharmaceutical preparations:

EXAMPLE A

Injection vials

A solution of 100 g of an active compound of the formula I and 5 g ofdisodium hydrogenphosphate is adjusted to pH 6.5 in 3 l ofdouble-distilled water using 2 N hydrochloric acid, sterile-filtered,dispensed into injection vials, lyophilized under sterile conditions andsealed aseptically. Each injection vial contains 5 mg of activecompound.

EXAMPLE B

Suppositories

A mixture of 20 g of an active compound of the formula I is fused with100 g of soya lecithin and 1400 g of cocoa butter, poured into mouldsand allowed to cool. Each suppository contains 20 mg of active compound.

EXAMPLE C

Solution

A solution of 1 g of an active compound of the formula I, 9.38 g ofNaH₂PO₄. 2 H₂O, 28.48 g of Na₂HPO₄. 12 H₂O and 0.1 g of benzalkoniumchloride in 940 ml of double-distilled water is prepared. The pH isadjusted to 6.8, and the solution is made up to 1 l and sterilized byirradiation. This solution can be used in the form of eye drops.

EXAMPLE D

Ointment

500 mg of an active compound of the formula I are mixed with 99.5 g ofpetroleum jelly under aseptic conditions.

EXAMPLE E

Tablets

A mixture of 1 kg of active compound of the formula I, 4 kg of lactose,1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearateis compressed to give tablets in the customary manner such that eachtablet contains 10 mg of active compound.

EXAMPLE F

Coated tablets

Analogously to Example E, tablets are pressed which are then coated in acustomary manner with a coating of sucrose, potato starch, talc,tragacanth and colorant.

EXAMPLE G

Capsules

2 kg of active compound of the formula I are dispensed into hardgelatine capsules in the customary manner such that each capsulecontains 20 mg of the active compound.

EXAMPLE H

Ampoules

A solution of 1 kg of active compound of the formula I in 60 1 ofdouble-distilled water is sterile-filtered, dispensed into ampoules,lyophilized under sterile conditions and aseptically sealed. Eachampoule contains 10 mg of active compound.

EXAMPLE I

Inhalation spray

14 g of active compound of the formula I are dissolved in 10 1 ofisotonic NaCl solution and the solution is dispensed into commerciallyavailable spray containers having a pump mechanism. The solution can besprayed into the mouth or nose. One burst of spray (approximately 0.1ml) corresponds to a dose of approximately 0.14 mg.

3 1 4 PRT Artificial Sequence Description of Artificial SequenceSynthetic cyclic peptide 1 Val Arg Gly Asp 1 2 4 PRT Artificial SequenceDescription of Artificial Sequence Synthetic cyclic peptide 2 Phe ArgGly Asp 1 3 4 PRT Artificial Sequence Description of Artificial SequenceSynthetic cyclic peptide 3 Gly Arg Gly Asp

What is claimed is:
 1. A compound of the formula Icyclo-(Arg-X-Asp-R¹)  I in which X is Gly, Ala or NH—NH—CO, where theamino acids mentioned can also be derivatized, and the amino acidresidues are linked to one another in a peptide-like manner via theα-carboxyl groups, R¹ is a radical of the formula II

 R²,R³,R⁴ in each case independently of one another are H, A, Ar, R⁵-Ar,Het or R⁵-Het, wherein if R⁴ is A, a methylene group present therein canbe replaced by N, O or S, A is alkyl having 1-6 C atoms, Ar is phenylwhich is unsubstituted or mono-, di- or trisubstituted by R⁷, R⁸ or R⁹,or is unsubstituted naphthyl, R⁵ is alkylene having 1-6 C atoms,R⁶,R^(6′) in each case, independently of one another, are H, A, benzylor phenyl, R⁷,R⁸,R⁹ in each case, independently of one another, are R⁶,OR⁶, Hal, NO₂, NR⁶R^(6′), NHCOR⁶, CN, NHSO₂R⁶, COOR⁶ or COR⁶, Hal is F,Cl, Br or I, and Het is a mono- or binuclear heterocycle having 1 to 4N, O, and/or S atoms, which can be unsubstituted or mono-, di- ortrisubstituted by Hal, NR⁶R^(6′), A, CN or N0₂, wherein optically activeamino acids and amino acid derivatives can be present in either their D-or L-forms, or a salt thereof.
 2. A compound according to claim 1,wherein said compound is in the form of an enantiomer.
 3. A compoundaccording to claim 1, wherein said compound is a)cyclo-(Arg-Gly-Asp-3-AMP-L-Phg) or a salt thereof; b)cyclo-(Arg-Gly-Asp-3-AMP-D-Phg) or a salt thereof; c)cyclo-(Arg-Gly-Asp-3-AMP-L-Val) (SEQ ID NO: 1) or a salt thereof; d)cyclo-(Arg-Gly-Asp-3-AMP-D-Val) or a salt thereof; e)cyclo-(Arg-Gly-Asp-3-AMP-Phe) (SEQ ID NO: 2) or a salt thereof; f)cyclo-(Arg-Gly-Asp-3-AMP-D-Phe) or a salt thereof; or g)cyclo-(Arg-Gly-Asp-3-AMP-Gly) (SEQ ID NO: 3) or a salt thereof.
 4. Aprocess for the preparation of a compound according to claim 1,comprising: a) treating a compound of formula III H—Z—OH  III  in whichZ is -Arg-X-Asp-R¹- —X-Asp-R¹-Arg- —Asp-R¹-Arg-X- or —R¹-Arg-X-Asp, or areactive derivative of a compound of the formula III with a cyclizingagent; or b) liberating a compound of formula I from one of itsfunctional derivatives by treatment with a solvolyzing orhydrogenolyzing agent; and/or converting a basic or acidic compound offormula I into one of its salts by treatment with an acid or base.
 5. Aprocess for production of a pharmaceutical preparation comprisingcombining at least one compound of the formula I according to claim 1 ora physiologically acceptable salt thereof with at least one solid,liquid or semiliquid excipient or auxiliary.
 6. A pharmaceuticalcomposition comprising at least one compound of the formula I accordingto claim 1 or a physiologically acceptable salt thereof and at least onesolid, liquid or semiliquid excipient or auxiliary.
 7. A method fortreating a patient suffering from thromboses, cardiac infarct, acoronary heart disorder, arteriosclerosis, a tumor, osteoporosis, aninflammation or an infection, comprising administering an effectiveamount of an intregrin inhibitor compound according to claim
 1. 8. Amethod of treating a patient suffering from a pathological conditionwhich is supported or propagated by angiogenesis, comprisingadministering an effective amount of a compound according to claim
 1. 9.A compound according to claim 1, wherein said compound is in the form ofa diastereomer.
 10. A compound according to claim 1, wherein A ismethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl ortert-butyl.
 11. A compound according to claim 1, wherein X is Gly or anN-methyl, N-ethyl, N-propyl, N-benzyl, or C_(α)-methyl derivativethereof; Ala or an N-methyl, N-ethyl, N-propyl, N-benzyl or C_(α)-methylderivative thereof; or NH—NH—CO.
 12. A compound according to claim 1,wherein R⁶ is H, methyl, ethyl, benzyl or phenyl.
 13. A compoundaccording to claim 1, wherein COR⁶ is formyl, acetyl, propionyl,butyryl, pentanoyl or hexanoyl.
 14. A compound according to claim 1,wherein R² and R³ are each independently H or A; R⁴ is H, A, Ar, R⁵-Ar,Het or R⁵-Het; and R⁶ and R^(6′) are each independently H or A.
 15. Acompound according to claim 1, wherein R² and R³ are each independentlyH or A; R⁴ is H, A, Ar, R⁵-Ar, Het or R⁵-Het; R⁶and R^(6′) are eachindependently H or A; and Ar is phenyl which is unsubstituted ormonosubstituted by R⁷.
 16. A compound according to claim 1, wherein R²and R³ are in each case, independently of one another, H or A; R⁴ is H,A, Ar, R⁵-Ar, Het or R⁵-Het; and R⁶ and R^(6′) are each independently Hor A; Ar is phenyl which is unsubstituted or monosubstituted by R⁷; andHet is a mononuclear aromatic or saturated heterocycle having 1 or 2 Nor O atoms, which can be unsubstituted or mono- or disubstituted by Hal,A, NR⁶R^(6′), CN or NO₂.
 17. A compound according to claim 1, wherein R²and R³ are in each case, independently of one another, H or A; R⁴ is H,A, Ar or R⁵-Ar; R⁶ and R^(6′) are H or A; and Ar is phenyl which isunsubstituted or monosubstituted by R⁷.
 18. A compound according toclaim 1, wherein X is Gly or Ala, R² and R³ are in each case,independently of one another, H or A; R⁴ is H, A, Ar or R⁵-Ar; R⁶ andR^(6′) are H or A; and Ar is phenyl which is unsubstituted ormonosubstituted by R⁷.
 19. A compound according to claim 1, whereinR²,R³ and R⁴ are each independently H, A, Ar, or R⁵-Ar, wherein if R⁴ isA, a methylene group therein can be replaced by N, O or S.
 20. Acompound according to claim 11, wherein R²,R³ and R⁴ are eachindependently H, A, Ar, or R⁵-Ar, wherein if R⁴ is A, a methylene grouptherein can be replaced by N, O or S.
 21. A pharmaceutical compositionaccording to claim 6, wherein said composition contains 0.5-100 mg of acompound of formula I or a physiologically acceptable salt thereof. 22.A method according to claim 7, wherein said compound is administered inan amount of 0.01-2 mg/kg of body weight per day.
 23. A pharmaceuticalcomposition preparation comprising at least one compound of the formulaI according to claim 11 or a physiologically acceptable salt thereof andat least one solid, liquid or semiliquid excipient or auxiliary.
 24. Amethod for treating a patient suffering from thromboses, cardiacinfarct, a coronary heart disorder, arteriosclerosis, a tumor,osteoporosis, an inflammation or an infection, comprising administeringan effective amount of an intregrin inhibitor compound according toclaim
 11. 25. A method of treating a patient suffering from apathological condition which is supported or propagated by angiogenesis,comprising administering an effective amount of a compound according toclaim
 11. 26. A pharmaceutical composition preparation comprising atleast one compound of the formula I according to claim 18 or aphysiologically acceptable salt thereof and at least one solid, liquidor semiliquid excipient or auxiliary.
 27. A method for treating apatient suffering from thromboses, cardiac infarct, a coronary heartdisorder, arteriosclerosis, a tumor, osteoporosis, an inflammation or aninfection, comprising administering an effective amount of an intregrininhibitor compound according to claim
 18. 28. A method of treating apatient suffering from a pathological condition which is supported orpropagated by angiogenesis, comprising administering an effective amountof a compound according to claim
 18. 29. A pharmaceutical compositionpreparation comprising at least one compound of the formula I accordingto claim 20 or a physiologically acceptable salt thereof and at leastone solid, liquid or semiliquid excipient or auxiliary.
 30. A method fortreating a patient suffering from thromboses, cardiac infarct, acoronary heart disorder, arteriosclerosis, a tumor, osteoporosis, aninflammation or an infection, comprising administering an effectiveamount of an intregrin inhibitor compound according to claim
 20. 31. Amethod of treating a patient suffering from a pathological conditionwhich is supported or propagated by angiogenesis, comprisingadministering an effective amount of a compound according to claim 20.